Haemaphysalis longicornis, which is widely distributed in China, can transmit various tick-borne diseases such as severe fever with thrombocytopenia syndrome, babesiosis, rickettsia disease and so on, and do great harm to human health and the development of animal husbandry. Chemical acaricides are the most traditional tick control method, but because of its many shortcomings, there is an urgent need to find a substitute with high efficiency, environmental protection and low toxicity. It has been found that some plant essential oils (EOs) have good insecticidal activity and environmental safety. In this study, the components of EOs from Pimenta racemosa and Eugenia caryophyllata were analyzed by gas chromatography-mass spectrometry (GC-MS), and their potential for application in the control of Haemaphysalis longicornis were studied. Gas chromatography-mass spectrometry analysis showed that the main components of P. racemosa EO were eugenol (64.07%), those of E. caryophyllata EO were Hexadecanoic acid, 2-methylpropyl ester (51.84%) and eugenol (39.76%). Larval packet test showed that the EOs of P. racemosa and E. caryophyllata had significant acaricidal activity against unfed larvae of H. longicornis, with LC50 values of 1.20 mg/mL and 0.47 mg/mL and LC90 values of 8.76 mg/mL and 2.91 mg/mL, respectively. The P. racemosa EO, E. caryophyllata EO and eugenol showed significant acaricidal activity against unfed nymph H. longicornis, with LC50 values of 1.65 mg/mL, 2.29 mg/mL and 0.93 mg/mL and LC90 values of 5.03 mg/mL, 11.01 mg/mL and 4.77 mg/mL, respectively. The P. racemosa EO, E. caryophyllata EO and eugenol showed significant acaricidal activity against unfed adults H. longicornis, with LC50 values of 0.51 mg/mL, 2.57 mg/mL and 1.83 mg/mL and LC90 values of 2.44 mg/mL, 11.44 mg/mL and 2.54 mg/mL, respectively. Enzyme assays revealed that the E. caryophyllata EO and eugenol significantly inhibited the activity of carboxylesterase (CarE), eugenol significantly inhibited the activity of catalase (CAT), and two EOs and eugenol had no significant effect on acetylcholinesterase (AchE) (p < 0.05). The above results suggest that the essential oils from P. racemosa and E. caryophyllata have great potential for use as alternatives to synthetic acaricides for tick control.

Nairobi Sheep Disease (NSD) is a typical tick-borne syndrome characterized by severe hemorrhagic gastroenteritis, spontaneous abortion, and a high case fatality rate in small ruminants. The pathogenic agent, Nairobi sheep disease virus (NSDV), has also been associated with human infections, indicating its possible zoonotic potential. Prior to this study, NSDV has been detected from ticks collected in Jilin, Hubei, and Liaoning provinces in China. In the present study, a total of 343 ticks (Haemaphysalis longicornis) were collected in Shandong province, China in 2020, and pooled into 16 libraries. Analysis of the meta-transcriptomic sequencing data identified NSDV strains SDWL07, SDWL08, and SDWL16 from three pools. The SDWL07 and SDWL16 strains were detected from unfed ticks, while SDWL08 was detected from cattle-feeding ticks. Phylogenetic analyses showed higher sequence identities between the three strains and other Chinese NSDV strains than those from India and Kenya. Phylogenetic analyses also revealed that they clustered together and fell within the China lineage, suggesting no potential genetic reassortment among them. In summary, this is the first report of the identification of NSDV in Shandong province, highlighting the continually expanding endemic regions of this pathogen. Surveillance of NSDV should be intensified in China, especially in areas where H. longicornis is endemic.

Haemaphysalis longicornis is a common tick species that carries several pathogens. There are few reports on the influence of different hosts on the structure of midgut microflora in H. longicornis. In this study, midgut contents of fully engorged female H. longicornis were collected from the surface of tiger (Panthera tigris) and deer (Dama dama). The bacterial genomic DNA of each sample was extracted, and the V3-V4 regions of the bacterial 16S rRNA were sequenced using the Illumina NovaSeq sequencing. The diversity of the bacterial community of the fully engorged female H. longicornis on the surface of tiger was higher than that of deer. In total, 8 phyla and 73 genera of bacteria annotations were detected in the two groups. At the phylum level, the bacterial phyla common to the two groups were Proteobacteria, Firmicutes, and Actinobacteriota. At the genus level, there were 20 common bacterial genera, among which the relative abundances of Coxiella, Morganella, Diplorickettsia, and Acinetobacter were high. The Morganella species was further identified to be Morganella morganii. The alpha diversity index indicated that the bacterial diversity of the tiger group was higher than that of the deer group. Bacteroidota, Patescibacteria, Desulfobacterota, Verrucomicrobiota, and Cyanobacteria were solely detected in the tiger group. A total of 52 bacterial genera were unique in the tiger group, while one bacterial genus was unique in the deer group. This study indicates that there are differences in the structure of the gut bacteria of the same tick species among different hosts. Further culture-based methods are needed to provide a more comprehensive understanding of the tick microbiota parasitizing different hosts.

BACKGROUND: Epigenetic modifications of histones play important roles in the response of eukaryotic organisms to environmental stress. However, many histone acetyltransferases (HATs), which are responsible for histone acetylation, and their roles in mediating the tick response to cold stress have yet to be identified. In the present study, HATs were molecularly characterized and their associations with the cold response of the tick Haemaphysalis longicornis explored.
METHODS: HATs were characterized by using polymerase chain reaction (PCR) based on published genome sequences, followed by multiple bioinformatic analyses. The differential expression of genes in H. longicornis under different cold treatment conditions was evaluated using reverse transcription quantitative PCR (RT-qPCR). RNA interference was used to explore the association of HATs with the cold response of H. longicornis.
RESULTS: Two HAT genes were identified in H. longicornis (Hl), a GCN5-related N-acetyltransferase (henceforth HlGNAT) and a type B histone acetyltransferase (henceforth HlHAT-B), which are respectively 960 base pairs (bp) and 1239 bp in length. Bioinformatics analysis revealed that HlGNAT and HlHAT-B are unstable hydrophilic proteins characterized by the presence of the acetyltransferase 16 domain and Hat1_N domain, respectively. RT-qPCR revealed that the expression of HlGNAT and HlHAT-B decreased after 3 days of cold treatment, but gradually increased with a longer period of cold treatment. The mortality rate following knockdown of HlGNAT or HlHAT-B by RNA interference, which was confirmed by RT-qPCR, significantly increased (P < 0.05) when H. longicornis was treated at the lowest lethal temperature (- 14 °C) for 2 h.
CONCLUSIONS: The findings demonstrate that HATs may play a crucial role in the cold response of H. longicornis. Thus further research is warranted to explore the mechanisms underlying the epigenetic regulation of the cold response in ticks.

Haemaphysalis longicornis, a three-host tick with a wide host range, is widely distributed in different countries and regions. It stands out among ticks due to its unique feature of having both parthenogenetic and bisexual populations. Despite their morphological resemblance, the characteristics of the parthenogenetic population have been overlooked. In this comprehensive study, we systematically compared the similarities and differences between these two populations. Our investigation revealed that the parthenogenetic H. longicornis, widely distributed in China, was found in ten provinces, surpassing the previously reported distribution. Notably, individuals from the parthenogenetic population exhibited a prolonged blood-feeding duration during the larval and nymph stages compared to their bisexual counterparts. Additionally, the life cycle of the parthenogenetic population was observed to be longer. A flow cytometry analysis indicated a DNA content ratio of approximately 2:3 between the bisexual and parthenogenetic populations. A phylogenetic analysis using whole mitochondrial genome sequences resulted in the separation of the phylogenetic tree into two distinct branches. A molecular analysis unveiled a consistent single T-base deletion at nucleotide 8497 in the parthenogenetic population compared to the bisexual population. Both populations displayed high viral infection capability and significant resistance to ivermectin. Intriguingly, despite these differences, the parthenogenetic population exhibited a similar life cycle to the bisexual population, retaining the ability to transmit pathogens such as Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland Virus (HRTV). These findings contribute to a deeper understanding of the distinct characteristics and similarities between different populations of H. longicornis, laying the foundation for future research in this field.

BACKGROUND: Ticks are blood-feeding ectoparasites with different host specificities and are capable of pathogen transmission. Iron regulatory proteins (IRPs) play crucial roles in iron homeostasis in vertebrates. However, their functions in ticks remain poorly understood. The aim of the present study was to investigate the characteristics, functions, molecular mechanisms, and the vaccine efficacy of IRP in the hard tick Haemaphysalis longicornis.
RESULTS: The full-length complementary DNA of IRP from Haemaphysalis longicornis (HlIRP) was 2973 bp, including a 2772 bp open reading frame. It is expressed throughout three developmental stages (larvae, nymphs, and adult females) and in various tissues (salivary glands, ovaries, midgut, and Malpighian tubules). Recombinant Haemaphysalis longicornis IRP (rHlIRP) was obtained via a prokaryotic expression system and exhibited aconitase, iron chelation, radical-scavenging, and hemolytic activities in vitro. RNA interference-mediated IRP knockdown reduced tick engorgement weight, ovary weight, egg mass weight, egg hatching rate, and ovary vitellin content, as well as prolonging the egg incubation period. Proteomics revealed that IRP may affect tick reproduction and development through proteasome pathway-associated, ribosomal, reproduction-related, and iron metabolism-related proteins. A trial on rabbits against adult Haemaphysalis longicornis infestation demonstrated that rHlIRP vaccine could significantly decrease engorged weight (by 10%), egg mass weight (by 16%) and eggs hatching rate (by 22%) of ticks. The overall immunization efficacy using rHlIRP against adult females was 41%.
CONCLUSIONS: IRP could limit reproduction and development in Haemaphysalis longicornis, and HlIRP was confirmed as a candidate vaccine antigen to impair tick iron metabolism and protect the host against tick infestation. © 2024 Society of Chemical Industry.

UNASSIGNED: Ticks are important blood-sucking ectoparasites that can transmit various pathogens, posing significant threats to the wellbeing of humans and livestock. Dabieshan tick virus (DBTV) was initially discovered in 2015 in Haemaphysalis longicornis ticks from the Dabieshan mountain region in Hubei Province, China. In recent years, DBTV has been discovered in various regions of China, including Shandong, Zhejiang, Liaoning, Hubei, Yunnan, and Guizhou Provinces. However, the researches on tick-borne transmission of DBTV are scarce.
UNASSIGNED: This study utilized the small RNA sequencing (sRNA-seq) method to identify tick-associated viruses in ticks collected from Chengde in Hebei Province and Yongcheng in Henan Province, leading to the discovery of a new DBTV strain in Hebei. The complete coding genome of DBTV Hebei strain was obtained through RNA-seq and Sanger sequencing. Furthermore, the transmission experiment of DBTV in H. longicornis was examined in laboratory for the first time.
UNASSIGNED: DBTV was detected in newly molted adult H. longicornis ticks collected in Chengde, Hebei Province. Additionally, DBTV was also detected in both unfed nymphs and engorged females of H. longicornis collected from Chengde, with a positive rate of 20% and 56.25%, respectively. The complete coding genome of DBTV (OP682840 and OP716696) were obtained, and phylogenetic analysis revealed that the DBTV Hebei strain clustered with previously reported DBTV strains. Furthermore, this virus was observed in engorged females, eggs, and larvae of the subsequent generation.
UNASSIGNED: It is necessary to expand the scope of DBTV investigation, particularly in northern China. This study demonstrated that DBTV can be transmitted from engorged females to larvae of the next generation. Moreover, the detection of DBTV in unfed nymphs and adults (which moulted from engorged nymphs) collected from the filed of Chengde suggests that H. longicornis serves as a potential transmission host and reservoir for DBTV through transstadial and transovarial transmission. However, there remains a lack of research on the isolation and pathogenicity of DBTV, highlighting the need for further studies to mitigate potential harm to the health of animals and humans.

BACKGROUND: Ticks serve as vectors for a diverse array of pathogens, including viruses responsible for both human and livestock diseases. Symbiotic bacteria hold significant potential for controlling tick-borne disease. However, the alteration of tick gut bacterial community in response to pathogen infection has not been analyzed for any tick-borne viruses. Here, the impact of severe fever with thrombocytopenia syndrome virus (SFTSV) infection on bacterial diversity in the gut of Haemaphysalis longicornis is investigated.
METHODS: Unfed tick females were artificially infected with SFTSV. The gut samples were collected and the genomic DNA was extracted. We then investigated alterations in gut bacterial composition in response to SFTSV infection through 16S rRNA gene sequencing.
RESULTS: The study found that a reduction in the number of operational taxonomic units (OTUs) in the tick gut following SFTSV infection. However, there were no significant changes in alpha diversity indices upon infection. Four genera, including Corynebacterium, Arthrobacter, Sphingomonas, and Escherichia, were identified as biomarkers for the tick gut without SFTSV infection. Notably, the predicted correlation network indicated that the biomarkers Sphingomonas and Escherichia exhibited positive correlations within the same subcommunity, which was altered upon viral infection.
CONCLUSIONS: These findings revealed that the change in tick gut bacterial composition upon SFTSV infection and could facilitate the discovery new target for tick-borne viral disease control.

Tick-borne Apicomplexan parasites pose a significant threat to both public health and animal husbandry. Identifying potential pathogenic parasites and gathering their epidemiological data are essential for prospectively preventing and controlling infections. In the present study, genomic DNA of ticks collected from two goat flocks (Goatflock1 and Goatflock2) and one dog group (Doggroup) were extracted and the 18S rRNA gene of Babesia/Theileria/Colpodella spp. was amplified by PCR and sequenced. Phylogenetic analysis was conducted based on the obtained sequences. The differences in pathogen positive rates between ticks of different groups were statistically analyzed using the Chi-square or continuity-adjusted Chi-square test. As a result, two pathogenic Theileria (T.) luwenshuni genotypes, one novel pathogenic Colpodella sp. HLJ genotype, and two potential novel Colpodella spp. (referred to as Colpodella sp. struthionis and Colpodella sp. yiyuansis in this study) were identified in the Haemaphysalis (H.) longicornis ticks. Ticks of Goatflock2 had a significantly higher positive rate of Colpodella spp. than those from Goatflock1 (χ2=92.10; P = 8.2 × 10-22) and Doggroup (χ2=42.34; P = 7.7 × 10-11), and a significantly higher positive rate of T. luwenshuni than Doggroup (χ2=5.38; P = 0.02). However, the positive rates of T. luwenshuni between Goatflock1 and Goatflock2 were not significantly different (χ2=2.02; P = 0.16), and so as the positive rates of both pathogens between Goatflock1 and Doggroup groups (P > 0.05). For either Colpodella spp. or T. luwenshuni, no significant difference was found in prevalence between male and female ticks. These findings underscore the potential importance of Colpodella spp. in domestic animal-attached ticks, as our study revealed two novel Colpodella spp. and identified Colpodella spp. in H. longicornis for the first time. The study also sheds light on goats\' potential roles in the transmission of Colpodella spp. to ticks and provides crucial epidemiological data of pathogenic Theileria and Colpodella. These data may help physicians, veterinarians, and public health officers prepare suitable detection and treatment methods and develop prevention and control strategies.

Haemaphysalis longicornis (the longhorned tick), the predominant tick species in China, serves as a vector for a variety of pathogens, and is capable of transmitting the tick-borne encephalitis virus (TBEV), the causative agent of tick-borne encephalitis. However, it is unclear how these ticks transmit TBEV. Langat virus (LGTV), which has a reduced pathogenicity in humans, has been used as a surrogate for TBEV. In this study, we aimed to investigate the vector competence of H. longicornis to transmit LGTV and demonstrate the efficient acquisition and transmission of LGTV between this tick species and mice. LGTV localization was detected in several tick tissues, such as the midgut, salivary glands, and synganglion, using quantitative PCR and immunohistochemical staining with a polyclonal antibody targeting the LGTV envelope protein. We demonstrated the horizontal transmission of LGTV to different developmental stages within the same generation but did not see evidence of vertical transmission. It was interesting to note that we observed mice acting as a bridge, facilitating the transmission of LGTV to neighboring naïve ticks during blood feeding. In conclusion, the virus-vector-host model employed in this study provides valuable insights into the replication and transmission of LGTV throughout its life cycle.